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The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

Abnormal GABAergic interneuron density, and imbalance of excitatory versus inhibitory tone, is thought to result in epilepsy, neurodevelopmental disorders, and psychiatric disease. Recent studies indicate that interneuron cortical density is determined primarily by the size of the precursor pool in the embryonic telencephalon. However, factors essential for regulating interneuron allocation from telencephalic multipotent precursors are poorly understood. Here we report that Olig1 represses production of GABAergic interneurons throughout the mouse brain. Olig1 deletion in mutant mice results in ectopic expression and upregulation of Dlx1/2 genes in the ventral medial ganglionic eminences and adjacent regions of the septum, resulting in an ∼30% increase in adult cortical interneuron numbers. We show that Olig1 directly represses the Dlx1/2 I12b intergenic enhancer and that Dlx1/2 functions genetically downstream of Olig1. These findings establish Olig1 as an essential repressor of Dlx1/2 and interneuron production in developing mammalian brain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Forward genetic screens in genetically accessible invertebrate organisms such as Drosophila melanogaster have shed light on transcription factors that specify formation of neurons in the vertebrate CNS. However, invertebrate models have, to date, been uninformative with respect to genes that specify formation of the vertebrate glial lineages. All recent insights into specification of vertebrate glia have come via monitoring the spatial and temporal expression patterns of individual transcription factors during development. In studies described here, we have taken this approach to the genome scale with an in silico screen of the Mahoney pictorial atlas of transcription factor expression in the developing CNS. From the population of 1445 known or probable transcription factors encoded in the mouse genome, we identify 12 novel transcription factors that are expressed in glial lineage progenitor cells. Entry-level screens for biological function establish one of these transcription factors, Klf15, as sufficient for genesis of precocious GFAP-positive astrocytes in spinal cord explants. Another transcription factor, Tcf4, plays an important role in maturation of oligodendrocyte progenitors.

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.

Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not to Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.

Progenitors within the ventral telencephalon can generate GABAergic neurons and oligodendrocytes, but regulation of the neuron-glial switch is poorly understood. We investigated the combinatorial expression and function of Dlx1&2, Olig2, and Mash1 transcription factors in the ventral telencephalon. We show that Dlx homeobox transcription factors, required for GABAergic interneuron production, repress oligodendrocyte precursor cell (OPC) formation by acting on a common progenitor to determine neuronal versus oligodendroglial cell fate acquisition. We demonstrate that Dlx1&2 negatively regulate Olig2-dependant OPC formation and that Mash1 promotes OPC formation by restricting the number of Dlx+ progenitors. Progenitors transplanted from Dlx1&2 mutant ventral telencephalon into newborn wild-type mice do not produce neurons but differentiate into myelinating oligodendrocytes that survive into adulthood. Our results identify another role for Dlx genes as modulators of neuron versus oligodendrocyte development in the ventral embryonic forebrain.

Disorders of cerebellar development can result in neurological disease and cancer. The identity of transcription factors that may uniquely mark and/or regulate development of single cerebellar cell types, however, is poorly understood. We used a library of approximately 1100 probes for expression of transcription factor (TF)-encoding genes (>70% of the mammalian 'transcriptome') to identify 227 genes with expression in developing neuronal and glial populations and 24 TFs that show cell-type- and stage-specific expression in granule cells, Purkinje cells and interneurons during postnatal cerebellar development. The utility of this panel is exemplified by analysis of medulloblastoma that shows upregulation of markers specific for early granule cell lineage, but not for other neuronal cell types, indicative of a unipotent precursor as well as a block in granule cell differentiation within the tumor. We propose that this atlas of the cerebellar transcriptome and the panel of 24 validated markers will be generally useful in analyses of mutations affecting postnatal cerebellar development and neoplasia.

Oligodendrocytes and their precursors serve critical roles in the maintenance of neurological function. Although activity of the transcription factors (TFs) Olig1, Olig2, Sox10, and Nkx2.2 is required during early oligodendrocyte development, their later expression in adult central nervous system is rather poorly characterized. Here we have analyzed co-expression patterns of these transcriptional proteins in the mouse cervical spinal cord. Our findings indicate that TF co-expression patterns describe heterogeneity in adult oligodendroglial populations (1) in distinct sub-regions of grey and white matter and (2) with respect to level of maturation from proliferating precursors to myelinating oligodendrocytes. Our findings suggest that TF co-expression patterns identify and might regulate distinct functional classes of grey and white matter oligodendroglia., (Copyright 2005 Wiley-Liss, Inc.)

Astrocytes are the most abundant and functionally diverse glial population in the vertebrate central nervous system (CNS). However, the mechanisms underlying astrocyte specification are poorly understood. It is well established that cellular diversification of neurons in the embryo is generated by position-dependent extrinsic signals and combinatorial interactions of transcription factors that direct specific cell fates by suppressing alternative fates. It is unknown whether a comparable process determines embryonic astrocyte identity. Indeed, astrocyte development is generally thought to take place in a position-independent manner. Here we show multiple functions of Stem cell leukaemia (Scl, also known as Tal1), which encodes a basic helix-loop-helix (bHLH) transcription factor, in the regulation of both astrocyte versus oligodendrocyte cell fate acquisition and V2b versus V2a interneuron cell fate acquisition in the p2 domain of the developing vertebrate spinal cord. Our findings demonstrate a regionally restricted transcriptional programme necessary for astrocyte and V2b interneuron development, with striking parallels to the involvement of SCL in haematopoiesis. They further indicate that acquisition of embryonic glial subtype identity might be regulated by genetic interactions between SCL and the transcription factor Olig2 in the ventral neural tube.

In the developing brain, transcription factors (TFs) direct the formation of a diverse array of neurons and glia. We identifed 1445 putative TFs in the mouse genome. We used in situ hybridization to map the expression of over 1000 of these TFs and TF-coregulator genes in the brains of developing mice. We found that 349 of these genes showed restricted expression patterns that were adequate to describe the anatomical organization of the brain. We provide a comprehensive inventory of murine TFs and their expression patterns in a searchable brain atlas database.

Olig1 and Olig2 are closely related basic helix-loop-helix (bHLH) transcription factors that are expressed in myelinating oligodendrocytes and their progenitor cells in the developing central nervous system (CNS). Olig2 is necessary for the specification of oligodendrocytes, but the biological functions of Olig1 during oligodendrocyte lineage development are poorly understood. We show here that Olig1 function in mice is required not to develop the brain but to repair it. Specifically, we demonstrate a genetic requirement for Olig1 in repairing the types of lesions that occur in patients with multiple sclerosis.

Epithelial-mesenchymal transformation is a critical developmental process reiterated in multiple organs throughout embryogenesis. Formation of endocardial cushions, primordia of valves and septa, is a classic example of epithelial-mesenchymal transformation. Several gene mutations are known to affect cardiac valve formation. Sox9 is activated when endocardial endothelial cells undergo mesenchymal transformation and migrate into an extracellular matrix, called cardiac jelly, to form endocardial cushions. In Sox9-null mutants, endocardial cushions are markedly hypoplastic. In these mutants, Nfatc1 is ectopically expressed and no longer restricted to endothelial cells. Further, Sox9-deficient endocardial mesenchymal cells fail to express ErbB3, which is required for endocardial cushion cell differentiation and proliferation. Our results reveal a succession of molecular steps in the pathway of endocardial cushion development. We propose that loss of Sox9 inhibits epithelial-mesenchymal transformation after delamination and initial migration, but before definitive mesenchymal transformation.

In developing neural tube, the basic helix-loop-helix (bHLH) transcription factor Olig2 interacts with the homeodomain transcription factor Nkx2.2 at two distinct stages. During neuronogenesis, a cross-repressive interaction appears to establish the precise boundary between the p3 and pMN domains. At later times, a cooperative interaction is noted because Nkx2.2 promotes maturation of oligodendrocyte progenitor cells specified by expression of Olig2. We show here that the Olig2 protein can form a physical complex with Nkx2.2 protein in mammalian cells and yeast two-hybrid trap assay. This interaction is specific because Olig2 does not bind to a biologically irrelevant homeodomain transcription factor (Nkx6.1), and Nkx2.2 does not interact with a biologically irrelevant bHLH protein (NeuroD). Deletion mapping analysis suggests that formation of an Olig2-Nkx2.2 physical complex is insufficient for the induction of oligodendrocyte progenitors in developing spine; however, the protein-protein interaction observed might be important for the cross-repressive interaction between Olig2 and Nkx2.2 that helps to establish the pMN-p3 boundary in the developing spinal cord.

Sox9 has essential roles in endochondral bone formation during axial and appendicular skeletogenesis. Sox9 is also expressed in neural crest cells, but its function in neural crest remains largely unknown. Because many craniofacial skeletal elements are derived from cranial neural crest (CNC) cells, we asked whether deletion of Sox9 in CNC cells by using the Cre recombinase/loxP recombination system would affect craniofacial development. Inactivation of Sox9 in neural crest resulted in a complete absence of cartilages and endochondral bones derived from the CNC. In contrast, all of the mesodermal skeletal elements and intramembranous bones were essentially conserved. The migration and the localization of Sox9-null mutant CNC cells were normal. Indeed, the size of branchial arches and the frontonasal mass of mutant embryos was comparable to that of WT embryos, and the pattern of expression of Ap2, a marker of migrating CNC cells, was normal. Moreover, in mouse embryo chimeras Sox9-null mutant cells migrated to their correct location in endochondral skeletal elements; however, Sox9-null CNC cells were unable to contribute chondrogenic mesenchymal condensations. In mutant embryos, ectopic expression of osteoblast marker genes, such as Runx2, Osterix, and Col1a1, was found in the locations where the nasal cartilages exist in WT embryos. These results indicate that inactivation of Sox9 causes CNC cells to lose their chondrogenic potential. We hypothesize that these cells change their cell fate and acquire the ability to differentiate into osteoblasts. We conclude that Sox9 is required for the determination of the chondrogenic lineage in CNC cells.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

Background: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons., Results: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region., Conclusions: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.

The Pax family of transcription factors plays important roles in vertebrate organogenesis. Pax-2 is a critical factor in the development of the mammalian urogenital system. Pax-2 is expressed in the epithelia of the ureter, the Müllerian duct, and the Wolffian duct and in the nephrogenic mesenchyme. Gene targeting in the mouse as well as natural mutations in mouse and man have demonstrated the requirement of Pax-2 in the development of these structures. Little is known about the molecular mechanisms regulating Pax-2 expression in the developing urogenital system. As a first step to reveal these mechanisms and to search for the elements and factors controlling Pax-2 expression we have characterized regulatory sequences of the Pax-2 gene in an in vivo reporter assay in the mouse. An 8.5-kb genomic region upstream of the Pax-2 transcription start site directed reporter gene activity in the epithelium of the pronephric duct at 8.25 days postcoitum (dpc) and in the Wolffian duct starting from 9.0 dpc. Expression in the Wolffian duct and its derivatives, the ureter, the collecting duct system, the seminal vesicles, the vas deferens, and the epididymis, was maintained at least until 18.5 dpc. Hence, an element(s) in the 8.5-kb upstream region is sufficient to initiate and maintain Pax-2 expression in the Wolffian duct and its derivatives. In order to more precisely map the Wolffian duct regulatory sequences, a deletion analysis of the 8.5-kb upstream region was performed in a transient in vivo reporter assay. A 0.4-kb subfragment was required for marker gene expression in the Wolffian duct. Misexpression of fgf8 under the control of the 8.5-kb upstream region resulted in polycystic kidneys, demonstrating the general usefulness of Pax-2 regulatory sequences in misexpression of foreign genes in the ureter and collecting duct system of the kidney in transgenic approaches in mice., (Copyright 2001 Academic Press.)

Expression of Pax-2 in the mouse gastrula is the first marker of the midbrain-hindbrain region. To address roles played by transcription factors in the process of neural plate pattern formation and to facilitate gain-of-function approaches in the study of midbrain-hindbrain and cerebellar development, we characterized regulatory sequences at the Pax-2 locus using an in vivo transgenic mouse reporter assay. An 8.5 kb fragment of genomic DNA located upstream of Pax-2 directed lacZ expression prior to neurulation (7.5 days post-coitum, dpc) in a region fated to become midbrain and hindbrain, and subsequently in developing neuroepithelium. While similar to the pattern of Pax-2 expression, reporter gene activity extended beyond the boundaries of Pax-2 expression, most probably reflecting purdurance of beta-galactosidase activity and an absence of DNA sequences that restrict Pax-2 expression to rhombomere 1 by 9. 5 dpc. In the fetal and neonatal brain, Pax-2-lacZ activity was confined largely to Purkinje cells and the external granule cell layer (EGL) of the cerebellum. A 4 kb regulatory element, in contrast, initiated neural expression at 8.25 dpc in the anterior hindbrain, but recapitulated all later aspects of Pax-2-lacZ activity observed with the larger transgene. These results indicate the presence of regulatory sequences upstream of the Pax-2 locus capable of directing gene expression in the developing midbrain, first rhombomere of the hindbrain, and its principal derivative, the cerebellum. Successful misexpression of Sonic hedgehog demonstrates that Pax-2 regulatory sequences should prove generally useful for transgenic gain-of-function approaches in mice.

In the Drosophila embryo, activation of wingless and engrailed in the parasegment requires paired, a member of the Pax family of transcription factors. We have explored the possible conservation of this regulatory hierarchy in the developing mouse brain. We find that Pax-2 is expressed prior to somite formation in the presumptive mid/hindbrain region. Shortly thereafter, Wnt-1 (the wingless orthologue) and Engrailed-1 are expressed in overlapping regions within the Pax-2 domain. Pax-5 expression commences later, at the 3-somite stage. Thus, the spatial and temporal expression of Pax-2 is consistent with a possible regulatory role in the activation of Wnt-1 and En-1.